PROJECT SUMMARY Extracellular matrix (ECM) is a complex network of macromolecules essential for proper functioning of cells in living tissues. Deregulation and disorganization of ECM lead to a wide spectrum of disorders, ranging from developmental abnormalities to metastatic cancers and fibrosis. Collagen is the major constituent of EMC and its timely and faithful secretion and deposition is critical for structural integrity and proper functioning of ECM. A variety of ECM-linked disorders is associated with aberrant secretion and deposition of collagen. Therefore, understanding how collagen secretion is regulated is at the heart of designing novel approaches to treat these disorders. Like many other secreted proteins, collagen is packaged into membrane-bound COPII vesicles at the endoplasmic reticulum (ER) exit sites and transported to the Golgi apparatus for further processing. While most COPII vesicles are 60 to 80 nm in diameter, 300 nm long unbendable procollagen fibers require large COPII vesicles for transport. Our group identified ubiquitylation ? a widespread post-translation modification ? as a key factor involved in collagen secretion. We showed that a vertebrate-specific ubiquitin ligase composed of the Cullin 3 (CUL3) scaffold and one of its substrate receptors KLHL12 (CUL3KLHL12) monoubiquitylates SEC31, one of the structural component of COPII vesicles, thereby allowing these vesicles to achieve large size suitable for packaging collagen. Recently, we identified a co-adaptor complex composed of two EF-hand- containing proteins, PEF1 and ALG2 that mediates calcium-dependent activation of CUL3KLHL12. The PEF1- ALG2 complex translates a transient rise in cytosolic calcium into persistent monoubiquitylation of SEC31, which, in turn, leads to biogenesis of large COPII vesicles and secretion of collagen. Strikingly, our in vitro assays show that in response to increasing calcium levels that activate CUL3KLHL12, unmodified PEF1 at the ligase is replaced with the monoubiquitylated protein, PEF1-UB. Monoubiquitylation of PEF1, therefore, plays an important role in activating CUL3KLHL12 ligase towards monoubiquitylation of SEC31 and thus also regulates large COPII biogenesis and collagen secretion. Our results identify the first calcium-dependent ubiquitin ligase as well as a rare example of protein interactions that depend on both calcium and ubiquitylation. However, the underlying mechanisms of CUL3KLHL12 regulation and how they are integrated into the pathway of collagen secretion have remained unknown. The goal of this proposal is to understand how reversible ubiquitylation of PEF1 and SEC31 is integrated into the regulatory network of large COPII vesicle formation and use this knowledge to correct the defects in collagen secretion observed in human diseases. I will begin by understanding how monoubiquitylation of PEF1 regulates CUL3KLHL12 activity. I will elucidate the mode of interaction of PEF1-UB with CUL3KLHL12 and the mechanism whereby PEF1-UB activates the ligase towards SEC31 monoubiquitylation. Since ubiquitylation is a reversible post-translational modification tightly regulated by deubiquitylating enzymes (DUBs), we hypothesized that deubiquitylation of PEF1-UB and SEC31- UB by dedicated DUBs constitutes an important regulatory factor in large COPII vesicle biogenesis. Our preliminary analyses identified USP9X as the DUB that acts directly on PEF1-UB. In this proposal, I will understand how the activity of this DUB is integrated into the regulatory network of CUL3KLHL12 activation and large COPII vesicle formation. I will use this knowledge to correct the defects in collagen secretion observed in human diseases. Since DUBs have recently gained increasing attention as targets of small molecule drugs, understanding how reversible ubiquitylation regulates collagen secretion will contribute significantly towards developing novel approaches to treat collagenopathies.